MECHANISM OF CALCIUM MOBILIZATION

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Cells possess various mechanisms for transducing information from the external environment to intracellular responses. Binding of ligands to surface receptors can lead to production of second messengers inside the cell and the first such messenger identified is cAMP. Receptor activation can also elevate Ca2+ due to mobilization of internal Ca2+ stores. The discovery of inositol trisphosphate (IP3) as a messenger for this process has centralized Ca2+ mobilization in signaling. Our research establishes that, in addition to IP3, the internal Ca2+ stores can be mobilized by two new messengers via totally independent pathways. Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) were discovered and their structures determined in my lab. CADPR is a new cyclic nucleotide derived from NAD, but unlike cAMP, its main signaling function is through direct modulation of Ca2+-induced CA2+ release (CICR), a major mechanism of Ca2+ mobilization beside the IP3-pathway. A variety of cells from plant to mammalian species are shown to be responsive to cADPR, indicating the generality of the signaling pathway. Similar to cADPR, NAADP, a metabolite of NADP, can also mobilize Ca2+ stores, but the release mechanism and the stores it acts on are distinct from cADPR. These two Ca2+ agonists are, nevertheless, intimately related, since the same metabolic enzymes can, under appropriate conditions, synthesize either one, suggesting a unified mechanism may regulate both pathways. Elucidation of the signaling pathways mediated by cADPR and NAADP is likely to have an important impact on our understanding of signal transduction mechanisms. Aim I is to develop and use a RIA for measuring cellular content of cADPR. Aim 2 is to purify and characterize a cGMP-dependent ADP-ribosyl cyclase. Aim 3 is to characterize the cGMP-dependent activation of the cyclase. These three Aims focus on identifying the stimuli and the activation mechanism of the cADPR-pathway. Results will provide the necessary evidence for establishing cADPR as a second messenger. Aim 4 is to determine the role of cADPR in the propagation of Ca2+ waves. It is generally believed that the CICR mechanism is crucial in propagating Ca2+ waves. Our finding that cADPR can modulate the Ca2+ sensitivity of CICR makes it relevant to investigate its role in the process. Aim 5 is to characterize the Ca2+ signaling mechanism mediated by NAADP. We will probe the structure-function relationship of NAADP, explore the regulatory mechanisms of its synthesis and investigate its possible role in mediating Ca2+ oscillations in cells.